This method is known from “Towards automated diffraction tomography: Part I-Data acquisition”, U. Kolb et al., Ultramicroscopy 107 (2007) 507-513.
There is great interest in determining the structure of macro-molecules, such as catalysts, proteins, viruses, DNA and RNA. The knowledge is of importance for understanding how e.g. proteins operate, how to produce, for example, more effective medicaments, enzymes, etc., and for example to understand why certain illnesses occur.
A group of techniques known as crystallography is used to determine the structure of molecules, of which X-ray crystallography is the most well-known. Here a multitude of diffraction patterns is recorded by irradiating a crystal by a beam of X-rays, and a diffraction pattern of said beam is recorded. A disadvantage of X-ray crystallography is that the size of the crystals must be rather large, e.g. 0.1 μm or more, because the interaction between the crystals and the X-ray beam is small. For many inorganic crystals this is not a problem, as these can easily be grown to a size of 0.1 μm or more, but it proves to be extremely difficult to grow crystals of e.g. proteins to such a size. X-ray diffraction is thus less suited for determining the structure of e.g. proteins.
The interaction between a beam of accelerated electrons, as used in e.g. an electron microscope, and the atoms of a crystal is much larger than when using X-rays. Therefore diffraction patterns of nano-crystals, with a diameter of less than 1 μm down to several nm, can be recorded with, for example, a transmission electron microscope (TEM).
In the known method described by U. Kolb, three-dimensional (3D) diffraction data are collected by manually tilting a crystal around a selected crystallographic axis and recording a set of diffraction patterns (a tilt series) at various crystallographic zones. In a second step, diffraction data from these zones are combined into a 3D data set and analyzed to yield the desired structure information. It is noted that data collection can be performed automatically. This involves a software module for a TEM enabling automated diffraction pattern collection while tilting around the goniometer axis. Kolb then proceeds to describe such a software module for a TEM, combining Scanning Transmission Electron Microscopy (STEM) imaging with diffraction pattern acquisition in nanodiffraction mode. It allows automated recording of diffraction tilt series from nanoparticles with a size down to 5 nm.
In the introduction Kolb teaches that the diffraction patterns can be recorded by illuminating the crystal with area selecting, the so-named Selected Area Electron Diffraction (SAED) technique, in which an aperture downstream of the diffraction plane is used to limit the part (the area) of the sample contributing to the diffraction pattern. The beam can be a convergent, focused beam (CBED), a substantially parallel beam, or any convergence angle therein between. Parallel illumination can be obtained by Köhler illumination. Alternatively a small aperture, known as the C2 aperture, can be used to decrease the beam diameter to a few nanometers while keeping the beam almost parallel. Kolb proceeds to describe that working in TEM mode with a small beam of typically 50 nm diameter makes it nearly impossible to position the beam with any degree of accuracy on a crystal that is larger than the beam. Therefore the position of the crystal is determined in STEM mode.
It is noted that Kolb mentions that in principle the diffraction patterns can be recorded using a more or less parallel beam, but fails to give an example of this. On the contrary, she proceeds showing Convergent Beam Electron Diffraction, and e.g. at page 509 of her article, lower right corner, says that the diffraction pattern is not focused in the back-focal plane.
A disadvantage of said method is that not all TEM's are equipped with a scanning unit, as a result of which not all TEM's can operate in STEM mode.
There is a need for a method that can be performed on an instrument that is not equipped with a scanning unit in order to operate in STEM mode.